309. Optimization of Dual AAV Vectors for Gene Therapy of Inherited Retinal Diseases

Abstract

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV limited cargo capacity prevents its application to therapies of inherited retinal diseases (IRD) due to mutations in genes over 5 kb. Dual AAV vectors, each containing one of the two halves of a large gene expression cassette, are emerging as promising tools to overcome this limitation. Dual AAV trans-splicing and hybrid vectors transduce efficiently the mouse and pig retina and are effective in animal models of IRD. However, some of dual AAV limitations include lower levels of transgene expression compared to a single AAV vector and the production of proteins shorter than expected from either the 5’- or 3’-half AAV. Thus, further development of dual AAV vectors is required before their clinical translation. To increase dual AAV recombination we have exploited various regions of homology while to mediate the degradation of the proteins shorter than expected we have tested the ability of various degradation signals. We found that the levels of transgene expression achieved with the alternative regions of homology are similar to those achieved with dual AAV vectors carrying the AK region of homology we have previously shown to be effective. Notably, we have identified a degradation signal which mediates the degradation of proteins shorter than expected from dual AAV vectors. In conclusion, our study outlines optimized features of dual AAV vectors that improve their safety and efficacy. This represents a step towards the clinical translation of dual AAV for retinal gene therapy.